Mutated anti-CD22 antibodies with increased affinity to CD22-expressing leukemia cells

ABSTRACT

Recombinant immunotoxins are fusion proteins composed of the Fv domains of antibodies fused to bacterial or plant toxins. RFB4 (Fv)-PE38 is an immunotoxin that targets CD22 expressed on B cells and B cell malignancies. The present invention provides antibodies and antibody fragments that have improved ability to bind the CD22 antigen of B cells and B cell malignancies compared to RFB4. Immunotoxins made with the antibodies and antibody fragments of the invention have improved cytotoxicity to CD22-expressing cancer cells. Compositions that incorporate these antibodies into chimeric immunotoxin molecules that can be used in medicaments and methods for inhibiting the growth and proliferation of leukemia and lymphoma cells.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.60/325,360, filed Sep. 26, 2001, the contents of which are herebyincorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable.

BACKGROUND OF THE INVENTION

Hematological malignancies are a major public health problem. It hasbeen estimated that in the year 2000, more than 50,000 new cases ofnon-Hodgkin's lymphoma and more than 30,000 new cases of leukemiaoccurred in the United States (Greenlee, R. T. et al., CA Cancer J.Clin., 50:7-33 (2000)) and more than 45,000 deaths were expected fromthese diseases. Many more patients live with chronic disease-relatedmorbidity. Unfortunately, in a high percentage of patients, conventionaltherapies are not able to induce long term complete remissions.

In the past several years immunotoxins have been developed as analternative therapeutic approach to treat these malignancies.Immunotoxins were originally composed of an antibody chemicallyconjugated to a plant or a bacterial toxin. The antibody binds to theantigen expressed on the target cell and the toxin is internalizedcausing cell death by arresting protein synthesis and inducing apoptosis(Brinkmann, U., Mol. Med. Today, 2:439-446 (1996)).

Hematological malignancies are an attractive target for immunotoxintherapies because tumor cells are easily accessible and the targetantigens are highly expressed (Kreitman, R. J. and Pastan, I., Semin.Cancer Biol., 6:297-306 (1995)). One of these antigens is CD25. Aclinical trial with immunotoxin LMB-2 (anti-Tac(Fv)PE38) that targetsCD25 showed that the agent was well tolerated and that it hadsubstantial anti-tumor activity (Kreitman, R. J. et al., Blood,94:3340-3348 (1999); Kreitman, R. J. et al., J. Clin. Oncol.,18:16222-1636 (2000)). A complete response was observed in one patientwith Hairy Cell Leukemia and partial responses were observed in patientswith Hairy Cell Leukemia, chronic lymphocytic leukemia, cutaneous T celllymphoma, Hodgkins disease and adult T cell leukemia.

Another antigen that has been used as an immunotoxin target is CD22, alineage-restricted B cell antigen expressed in 60-70% of B celllymphomas and leukemias. CD22 is not present on the cell surface in theearly stages of B cell development and is not expressed on stem cells(Tedder, T. F. et al., Annu. Rev. Immunol., 5:481-504 (1997)). Clinicaltrials have been conducted with an immunotoxin containing an anti-CD22antibody, RFB4, or is Fab fragment, coupled to deglycosylated ricin A.In these trials, substantial clinical responses have been observed;however, severe and in certain cases fatal, vascular leak syndrome wasdose limiting (Sausville, E. A. et al., Blood, 85:3457-3465 (1995);Amlot, P. L. et al., Blood, 82:2624-2633 (1993); Vitetta, E. S. et al.,Cancer Res., 51:4052-4058 (1991)).

As an alternative approach, the RFB4 antibody was used to make arecombinant immunotoxin in which the Fv fragment in a single chain formis fused to a 38 kDa truncated form of Pseudomonas exotoxin A (PE38).PE38 contains the translocating and ADP ribosylating domains of PE butnot the cell-binding portion (Hwang, J. et al., Cell, 48:129-136(1987)). RFB4 (Fv)-PE38 is cytotoxic towards CD22-positive cells(Mansfield, E. et al., Biochem. Soc. Trans., 25:709-714 (1997)). Tostabilize the single chain Fv immunotoxin and to make it more suitablefor clinical development, cysteine residues were engineered intoframework regions of the V_(H) and V_(L) (Mansfield, E. et al., Blood,90:2020-2026 (1997)) generating the molecule RFB4 (dsFv)-PE38.

RFB4 (dsFv)-PE38 is able to kill leukemic cells from patients andinduced complete remissions in mice bearing lymphoma xenografts(Kreitman, R. J. et al., Clin. Cancer Res., 6:1476-1487 (2000);Kreitman, R. J. et al., Int. J. Cancer, 81:148-155 (1999)). RFB4(dsFv)-PE38 (BL22) is currently being evaluated in a phase I clinicaltrial at the National Cancer Institute in patients with hematologicalmalignancies. Sixteen patients with purine analogue resistant hairy cellleukemia were treated with BL22 and 11 (86%) have achieved completeremissions (Kreitman, R. J. et al., N. Engl. J. Med. (2001)).

Because of the clinical benefits obtained with BL22, and becauseimproved binding affinity has been shown to improve selective tumordelivery of scFvs (Adams et al., Cancer Res. 58:485-490 (1998)),improving the binding affinity of scFvs and other targeting moieties(such as dsFvs, Fabs. and F(ab′)₂) of immunoconjugates could improve theefficiency of these agents in delivering effector molecules to malignantB-cells. Improved targeting would likely decrease the dose necessary toachieve complete remission of these cancers.

The factors that influence binding affinity are multifaceted andobtaining mutant scFvs with improved affinity is not trivial. Althoughantibody-antigen crystal structure can suggest which residues areinvolved in binding, but atomic resolution structural data are notavailable for most antibodies. Moreover, even when such data isavailable it cannot generally be predicted which residues and whichmutations will result in an antibody with increased antigen bindingactivity.

BRIEF SUMMARY OF THE INVENTION

The present invention provides improved antibodies for binding toCD22-expressing cells (a “CD22+” cell), especially cancer cells thatexpress CD22 on their exterior surface (a “CD22+ cancer cell”). In thisregard, the invention provides anti-CD22 antibodies with a variablelight (V_(L)) chain having the sequence of antibody RFB4 and a variableheavy (V_(H)) chain having the sequence of antibody RFB4, but in whichresidues 100, 100A and 100B of CDR3 of said V_(H) chain (as numbered bythe Kabat and Wu numbering system) have an amino acid sequence selectedfrom the group consisting of: THW, YNW, TTW, and STY. The antibody canbe a full length antibody molecule, but is preferably a single chain Fv(“scFv”), a disulfide stabilized Fv (“dsFv”), an Fab, or an F(ab′). In aparticularly preferred form, the antibody is a dsFv. (For convenience ofreference, the term “antibody” in the text below refers to full lengthantibodies and, more preferably, to scFv, dsFv, Fab, or F(ab′)).

The invention further provides compositions comprising one of theseantibodies conjugated or fused to a therapeutic moiety or a detectablelabel. The therapeutic moiety can be a cytotoxin, a drug, aradioisotope, or a liposome loaded with a drug or a cytotoxin. Inpreferred embodiments, the effector moiety is a cytotoxin. The cytotoxincan be selected from the group consisting of ricin A, abrin, ribotoxin,ribonuclease, saporin, calicheamycin, diphtheria toxin or a cytotoxicsubunit or mutant thereof, a Pseudomonas exotoxin, a cytotoxic portionthereof, a mutated Pseudomonas exotoxin, a cytotoxic portion thereof,and botulinum toxins A through F. In preferred forms, the cytotoxin is aPseudomonas exotoxin or cytotoxic fragment thereof, or a mutatedPseudomonas exotoxin or a cytotoxic fragment thereof. In particularlypreferred forms, the Pseudomonas exotoxin is selected from the groupconsisting of PE35, PE38, PE38 KDEL, PE40, PE4E, and PE38QQR. In themost preferred embodiment, the Pseudomonas exotoxin is PE38. Thecompositions may further comprise a pharmaceutically acceptable carrier.

The invention further provides the use of an anti-CD22 antibody with avariable light (V_(L)) chain having the sequence of antibody RFB4 and avariable heavy (V_(H)) chain having the sequence of antibody RFB4,provided that residues 100, 100A and 100B of CDR3 of said V_(H) chainhave an amino acid sequence selected from the group consisting of: THW,YNW, TTW, and STY, for the manufacture of a medicament to inhibit thegrowth of a CD22+ cancer cell. The antibody can be, for example, a fulllength antibody, an scFv, dsFv, a Fab, or a F(ab′)₂. In a particularlypreferred form, the antibody is a dsFv. The invention further providesfor the use of a composition for the manufacture of a medicament forinhibiting growth of a CD22+ cancer cell, which composition comprises anantibody as just described conjugated or fused to a therapeutic moietyor a detectable label. The therapeutic moiety can be, for example, acytotoxin, a drug, a radioisotope, or a liposome loaded with a drug or acytotoxin. In preferred forms, the therapeutic moiety is a cytotoxin.The cytotoxin is preferably selected from the group consisting of ricinA, abrin, ribotoxin, ribonuclease, saporin, calicheamycin, diphtheriatoxin or a cytotoxic subunit or mutant thereof, a Pseudomonas exotoxin,a cytotoxic portion thereof, a mutated Pseudomonas exotoxin, a cytotoxicportion thereof, and botulinum toxins A through F. In preferred uses,the cytotoxin is a Pseudomonas exotoxin or cytotoxic fragment thereof,or a mutated Pseudomonas exotoxin or a cytotoxic fragment thereof and,in particularly preferred uses, is selected from the group consisting ofPE35, PE38, PE38 KDEL, PE40, PE4E, and PE38QQR, with PE38 being the mostpreferred.

In another group of embodiments, the invention provides nucleic acidsencoding anti-CD22 antibodies with a variable light (V_(L)) chain havingthe sequence of antibody RFB4 and a variable heavy (V_(H)) chain havingthe sequence of antibody RFB4, in which residues 100, 100A and 100B ofCDR3 of said V_(H) chain have an amino acid sequence selected from thegroup consisting of: THW, YNW, TTW, and STY. The antibody can, forexample, be a full-length antibody, or can be selected from the groupconsisting of an scFv, a dsFv, a Fab, or a F(ab′)₂. In particularlypreferred forms, the antibody is a dsFv. The nucleic acid can furtherencode a polypeptide which is a therapeutic moiety or a detectablelabel. The therapeutic moiety can be a drug or a cytotoxin. Thecytotoxin can be, for example, a Pseudomonas exotoxin or cytotoxicfragment thereof, or a mutated Pseudomonas exotoxin or a cytotoxicfragment thereof and is preferably selected from the group consisting ofPE35, PE38, PE38 KDEL, PE40, PE4E, and PE38QQR. In the most preferredform, the Pseudomonas exotoxin is PE38. The invention further providesexpression vectors comprising any of the nucleic acids described aboveoperably linked to a promoter.

In yet another group of embodiments, the invention provides methods ofinhibiting growth of a CD22+ cancer cell. The methods comprisecontacting the cell with an anti-CD22 antibody with a variable light(V_(L)) chain having the sequence of antibody RFB4 and a variable heavy(V_(H)) chain having the sequence of antibody RFB4, provided thatresidues 100, 100A and 100B of CDR3 of said V_(H) chain have an aminoacid sequence selected from the group consisting of: THW, YNW, TTW, andSTY, which antibody is fused or conjugated to a therapeutic moiety,which therapeutic moiety inhibits growth of said cell. The antibody canbe an scFv, a dsFv, a Fab, or a F(ab′)₂. In a particularly preferredform, the antibody is a dsFv. The therapeutic moiety can be, forexample, a cytotoxin, a drug, a radioisotope, or a liposome loaded witha drug or a cytotoxin. In preferred forms, the therapeutic moiety is acytotoxin. The cytotoxin can be, for example, ricin A, abrin, ribotoxin,ribonuclease, saporin, calicheamycin, diphtheria toxin or a cytotoxicsubunit or mutant thereof, a Pseudomonas exotoxin, a cytotoxic portionthereof, a mutated Pseudomonas exotoxin, a cytotoxic portion thereof,and botulinum toxins A through F. In preferred forms, the cytotoxin is aPseudomonas exotoxin or cytotoxic fragment thereof, or a mutatedPseudomonas exotoxin or a cytotoxic fragment thereof. In particularlypreferred embodiments, the Pseudomonas exotoxin is selected from thegroup consisting of PE35, PE38, PE38 KDEL, PE40, PE4E, and PE38QQR. Inthe most preferred embodiment, the Pseudomonas exotoxin is PE38.

The invention further provides methods for detecting the presence of aCD22+ cancer cell in a biological sample, said method comprisingcontacting cells of said biological sample with an anti-CD22 antibodywith a variable light (V_(L)) chain having the sequence of a V_(L) chainof antibody RFB4 and a variable heavy (V_(H)) chain having the sequenceof a V_(H) chain antibody RFB4, provided that residues 100, 100A and100B of CDR3 of the V_(H) chain of said anti-CD22 antibody have an aminoacid sequence selected from the group consisting of: THW, YNW, TTW, andSTY, said antibody being fused or conjugated to a detectable label; anddetecting the presence or absence of said label, wherein detecting thepresence of said label indicates the presence of a CD22+ cancer cell insaid sample. The antibody can be, for example, selected from the groupconsisting of an scFv, a dsFv, a Fab, or a F(ab′)₂. In a particularlypreferred form, the antibody is a dsFv.

In another group of embodiments, the invention provides kits fordetecting the presence of a CD22+ cancer cell in a biological sample,said kit comprising a container, and an anti-CD22 antibody with avariable light (V_(L)) chain having the sequence of a V_(L) chain ofantibody RFB4 and a variable heavy (V_(H)) chain having the sequence ofa V_(H) chain antibody RFB4, provided that residues 100, 100A and 100Bof CDR3 of the V_(H) chain of said anti-CD22 antibody have an amino acidsequence selected from the group consisting of: THW, YNW, TTW, and STYwhich antibody is fused or conjugated to a detectable label. In someembodiments, the antibody is selected from the group consisting of anscFv, a dsFv, a Fab, or a F(ab′)₂.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 sets forth the nucleotide sequence (SEQ ID NO:3) andamino acid sequence (SEQ ID NO:4) of the variable region of the RFB4light chain and the nucleotide sequence (SEQ ID NO:1) and amino acidsequence (SEQ ID NO:2) of the variable region of the RFB4 heavy chain.

FIG. 2. FIG. 2 is a print out of Entry Number 038145 of the Kabatdatabase showing the amino acid sequence (SEQ NO:4) of the variableregion of the RFB4 light chain and the Kabat position numberingcorresponding to each amino acid residue.

FIG. 3. FIG. 3 is a print out of Entry Number 038146 of the Kabatdatabase showing the amino acid sequence (SEQ NO:2) of the variableregion of the RFB4 heavy chain and the Kabat position numberingcorresponding to each amino acid residue.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides antibodies and antibody fragments thathave increased binding affinity for cancer cells bearing the CD22antigen compared to the anti-CD22 antibody known in the art as RFB4.Mutated scFvs have been discovered and isolated that have increases inaffinity of from 3.5 to 15-fold the affinity of wild-type RFB4.Immunotoxins made with these high affinity variants had a significantincrease in cytotoxic activity compared to a like immunotoxin made withwild-type RFB4.

These mutants change the amino acid sequence of the residues atpositions 100, 100A, and 100B of CDR3 of the V_(H) chain of RFB4 fromthe wild type sequence SSY to THW, YNW, or STY. A single amino acidchange, for example, the one amino acid difference between the sequenceSSY and STY, reduced the dissociation constant (K_(D)) of a chimericimmunotoxin made with the resulting scFV to 49 ID, compared to the 85 kDof a like immunotoxin using the parental antibody RFB4 sequence. Achange of SSY to TTW lowered the kD of the resulting immunotoxin to 24kD. Even more impressively, the mutation of the residues SSY to YNWimproved the affinity of the resulting immunotoxin from the 85 kD of theimmunotoxin employing the parental, wild-type RFB4 antibody to 10 kD.And, substituting THW for the wild-type sequence of SSY improved theaffinity even more, to 6 kD.

These improved affinities are reflected in improved cytotoxic activityof immunotoxins made by fusing or conjugating the antibodies orfragments thereof which retain antigen recognition ability to acytotoxin. For example, tests of an exemplar immunotoxin made fromcombining an scFv having an RFB4 V_(H) CDR3 sequence in which SSY wasmutated to STY to a cytotoxin showed that the amount of the immunotoxinneeded to inhibit 50% of the protein synthesis (known as the IC₅₀ of theimmunotoxin) in CD22-expressing cancer cells from patients was reducedby as much as much as 7-fold compared to a like immunotoxin made withthe wild-type SSY sequence. Similar tests showed with an immunotoxinmade with the THW sequence showed that the THW sequence increased thecytotoxic activity of the immunotoxin to cells of the CD22-bearingcancer chronic lymphocytic leukemia by 50 times. An immunotoxin was alsomade with a dsFv having the THW sequence and tested for cytotoxicityagainst cells from patients having chronic lymphocytic leukemia (CLL) orhairy cell leukemia (HCL). The THW dsFv immunotoxin showed 10 to 40times higher cytotoxicity against CLL cells than did the wild type RFB4dsFv immunotoxin, and 4 to 7 times higher cytotoxicity against HCL cellsthan the wild type RFB4 dsFv immunotoxin.

The improved affinity of the improved antibody and antibody fragmentsprovided by the present invention can be incorporated into chimericimmunoconjugates to improve the ability of the chimeric immunoconjugateto target B-cells bearing the CD22 antigen. The immunoconjugates can,for example, bear a detectable label such as a radioisotope or areporter enzyme. These labeled immunoconjugates be used, for example, inin vitro assays to detect the presence of CD22-expressing cells in abiological sample. Typically, the biological sample will be a bloodsample or lymphocytes from a blood sample.

In another set of in vitro uses, the immunoconjugate bears a cytotoxinrather than a detectable label. Such immunotoxins can be used to purge ablood sample or culture of lymphocytes from a patient. The purged sampleor culture can then be readministered to the patient to boost thefunctional white-blood cell population.

In in vivo uses, immunotoxins made with the antibodies or antibodyfragments of the invention can be used to inhibit the growth andproliferation of cancer cells bearing the CD22 antigen. As noted in theBackground section, an immunotoxin made with the parental antibody,RFB4, is currently in human clinical trials and, when tested against anexemplar CD22-expressing cancer, caused complete remissions in 86% ofthe patients. The greater affinity of the antibodies and antibodyfragments of the invention compared to the parental antibody, RFB4, andthe greater cytotoxicity of the resulting immunotoxins means thatsmaller amounts of the immunotoxins can be administered, therebyachieving the same therapeutic effect while reducing the chance of sideeffects.

In preferred embodiments, the antibody is a scFv or a dsFv. Many of therecombinant immunotoxins produced from constructs of scFv are one-thirdthe size of IgG-toxin chemical conjugates and are homogeneous incomposition. Elimination of the constant portion of the IgG moleculefrom the scFv results in faster clearance of the immunotoxin afterinjection into animals, including primates, and the smaller size of theconjugates improves drug penetration in solid tumors. Together, theseproperties lessen the side effects associated with the toxic moiety byreducing the time in which the immunotoxin (IT) interacts withnon-target tissues and tissues that express very low levels of antigen.Making disulfide stabilized Fvs (dsFvs) from anti-CD22 antibodies isdiscussed in the co-owned application of FitzGerald et al.,International Publication Number WO 98/41641, which is incorporatedherein by reference.

These advantages, however, are offset to some degree by the loss ofantigen binding affinity that occurs when IgGs are converted to scFvs(Reiter et al., Nature Biotechnol. 14:239-1245 (1996)). Increasingaffinity has been shown to improve selective tumor delivery of scFvs(Adams et al., Cancer Res. 58:485-490 (1998)), and is likely to increasetheir usefulness in tumor imaging and treatment. Therefore, increasingthe affinity of scFvs and other targeting moieties (such as dsFvs, Fabs.and F(ab′)₂ of immunoconjugates is desirable to improve the efficiencyof these agents in delivering effector molecules, such as toxins andother therapeutic agents, to their intended targets. The improvedaffinity of the antibodies of the invention therefore is an importantadvance in the delivery of toxins, drugs, and other therapeutic agentsto cell of CD22-expressing cancers.

In the sections below, the terms used herein are defined for additionalclarity. The invention is described in more detail. Finally, theexamples demonstrate the construction and testing of exemplaryimmunotoxins using antibodies in which STY, YNW, TTW, or THW wassubstituted for the SSY sequence of the RFB4 antibody.

Definitions

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

“CD22” refers to a lineage-restricted B cell antigen belonging to the Igsuperfamily. It is expressed in 60-70% of B cell lymphomas and leukemiasand is not present on the cell surface in early stages of B celldevelopment or on stem cells. See, e.g. Vaickus et al., Crit. Rev.Oncol/Hematol. 11:267-297 (1991).

As used herein, the term “anti-CD22” in reference to an antibody, refersto an antibody that specifically binds CD22 and includes reference to anantibody which is generated against CD22. In preferred embodiments, theCD22 is a primate CD22 such as human CD22. In a particularly preferredembodiment, the antibody is generated against human CD22 synthesized bya non-primate mammal after introduction into the animal of cDNA whichencodes human CD22.

“RFB4” refers to a mouse IgG1 monoclonal antibody that specificallybinds to human CD22. RFB4 is commercially available under the name RFB4from several sources, such as Southern Biotechnology Associates, Inc.(Birmingham Ala.; Cat. No. 9360-01) and Autogen Bioclear UK Ltd. (Caine,Wilts, UK; Cat. No. AB147). RFB4 is highly specific for cells of the Blineage and has no detectable cross-reactivity with other normal celltypes. Li et al., Cell. Immunol. 118:85-99 (1989). The heavy and lightchains of RFB4 have been cloned. See, Mansfield et al., Blood90:2020-2026 (1997), which is incorporated herein by reference. Thenucleotide sequence and amino acid sequences of the RFB4 heavy chain areSEQ ID NO:1 and SEQ ID NO:2, respectively. The nucleotide sequence andamino acid sequences of the RFB4 light chain are SEQ ID NO:3 and SEQ IDNO:4, respectively. The sequences are set forth in FIG. 1.

Unless otherwise indicated, references herein to amino acid positions ofthe RFB4 heavy or light chain refer to the numbering of the amino acidsunder the “Kabat and Wu” system. See, Kabat, E., et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, U.S. Government Printing Office, NIHPublication No. 91-3242 (1991), which is hereby incorporated byreference system. It should be noted that the number accorded to aresidue under the Kabat and Wu system does not necessarily correspond tothe number that one might obtain for a residue in a given heavy or lightchain by counting from the amino terminus of that chain. FIGS. 2 and 3show the correlation between the sequential numbering of the residues ofthe RFB4 light and heavy chains and the Kabat and Wu numbering of thoseresidues. For convenience, the “Kabat and Wu” numbering is sometimesreferred to herein as “Kabat” numbering.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies), heteroconjugate antibodies (e.g.,bispecific antibodies), recombinant single chain Fv fragments (scFv),and disulfide stabilized (dsFv) Fv fragments (see, co-owned U.S. Pat.No. 5,747,654, which is incorporated herein by reference). The term“antibody” also includes antigen binding forms of antibodies (e.g.,Fab′, F(ab′)₂, Fab, Fv and rigG. See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.); Goldsby et al., eds.,Kuby, J., Immunology, 4th Ed., W.H. Freeman & Co., New York (2000).

An antibody immunologically reactive with a particular antigen can begenerated by recombinant nethods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, see, e.g., Huse, etal., Science 246:1275-1281 (1989); Ward, et al., Nature 341:544-546(1989); and Vaughan, et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region, (theregions are also known as “domains”). Light and heavy chain variableregions contain a “framework” region interrupted by three hypervariableregions, also called “complementarity-determining regions” or “CDRs”.The extent of the framework region and CDRs have been defined. See,Kabat and Wu, supra. The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

References to “V_(H)” or a “VH” refer to the variable region of animmunoglobulin heavy chain, including an Fv, scFv, dsFv or Fab.References to “V_(L)” or a “VL” refer to the variable region of animmunoglobulin light chain, including of an Fv, scFv, dsFv or Fab.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site.

The term “linker peptide” includes reference to a peptide within anantibody binding fragment (e.g., Fv fragment) which serves to indirectlybond the variable domain of the heavy chain to the variable domain ofthe light chain.

The term “parental antibody” means any antibody of interest which is tobe mutated or varied to obtain antibodies or fragments thereof whichbind to the same epitope as the parental antibody, but with higheraffinity.

The term “hotspot” means a portion of a nucleotide sequence of a CDR orof a framework region of a variable domain which is a site ofparticularly high natural variation. Although CDRs are themselvesconsidered to be regions of hypervariability, it has been learned thatmutations are not evenly distributed throughout the CDRs. Particularsites, or hotspots, have-been identified as these locations whichundergo concentrated mutations. The hotspots are characterized by anumber of structural features and sequences. These “hotspot motifs” canbe used to identify hotspots. Two consensus sequences motifs which areespecially well characterized are the tetranucleotide sequence RGYW andthe serine sequence AGY, where R is A or G, Y is C or T, and W is A orT.

A “targeting moiety” is the portion of an immunoconjugate intended totarget the immunoconjugate to a cell of interest. Typically, thetargeting moiety is an antibody, a scFv, a dsFv, an Fab, or an F(ab′)₂.

A “toxic moiety” is the portion of a immunotoxin which renders theimmunotoxin cytotoxic to cells of interest.

A “therapeutic moiety” is the portion of an immunoconjugate intended toact as a therapeutic agent.

The term “therapeutic agent” includes any number of compounds currentlyknown or later developed to act as anti-neoplastics,anti-inflammatories, cytokines, anti-infectives, enzyme activators orinhibitors, allosteric modifiers, antibiotics or other agentsadministered to induce a desired therapeutic effect in a patient. Thetherapeutic agent may also be a toxin or a radioisotope, where thetherapeutic effect intended is, for example, the killing of a cancercell.

A “detectable label” means, with respect to an immunoconjugate, aportion of the immunoconjugate which has a property rendering itspresence detectable. For example, the immunoconjugate may be labeledwith a radioactive isotope which permits cells in which theimmunoconjugate is present to be detected in immunohistochemical assays.

The term “effector moiety” means the portion of an immunoconjugateintended to have an effect on a cell targeted by the targeting moiety orto identify the presence of the immunoconjugate. Thus, the effectormoiety can be, for example, a therapeutic moiety, a toxin, a radiolabel,or a fluorescent label.

The term “immunoconjugate” includes reference to a covalent linkage ofan effector molecule to an antibody. The effector molecule can be animmunotoxin.

The terms “effective amount” or “amount effective to” or“therapeutically effective amount” includes reference to a dosage of atherapeutic agent sufficient to produce a desired result, such asinhibiting cell protein synthesis by at least 50%, or killing the cell.

The term “toxin” includes reference to abrin, ricin, Pseudomonasexotoxin (PE), diphtheria toxin (DT), botulinum toxin, or modifiedtoxins thereof. For example, PE and DT are highly toxic compounds thattypically bring about death through liver toxicity. PE and DT, however,can be modified into a form for use as an immunotoxin by removing thenative targeting component of the toxin (e.g., domain Ia of PE or the Bchain of DT) and replacing it with a different targeting moiety, such asan antibody.

The term “contacting” includes reference to placement in direct physicalassociation.

An “expression plasmid” comprises a nucleotide sequence encoding amolecule or interest, which is operably linked to a promoter.

As used herein, “polypeptide”, “peptide” and “protein” are usedinterchangeably and include reference to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers. The terms also apply to polymers containing conservativeamino acid substitutions such that the protein remains functional.

The term “residue” or “amino acid residue” or “amino acid” includesreference to an amino acid that is incorporated into a protein,polypeptide, or peptide (collectively “peptide”). The amino acid can bea naturally occurring amino acid and, unless otherwise limited, canencompass known analogs of natural amino acids that can function in asimilar manner as naturally occurring amino acids.

The amino acids and analogs referred to herein are described byshorthand designations as follows in Table A:

TABLE A Amino Acid Nomenclature Name 3-letter 1-letter Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys CGlutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His HHomoserine Hse — Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Methionine sulfoxide Met (O) — Methionine Met (S—Me) —methylsulfonium Norleucine Nle — Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

“conservative substitution”, when describing a protein refers to achange in the amino acid composition of the protein that does notsubstantially alter the protein's activity. Thus, “conservativelymodified variations” of a particular amino acid sequence refers to aminoacid substitutions of those amino acids that are not critical forprotein activity or substitution of amino acids with other amino acidshaving similar properties (e.g., acidic, basic, positively or negativelycharged, polar or non-polar, etc.) such that the substitutions of evencritical amino acids do not substantially alter activity. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. The following six groups in Table B each contain aminoacids that are conservative substitutions for one another:

TABLE B 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D),Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R),Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also,Creighton, PROTEINS, W. H. Freeman and Company, New York (1984).

The terms “substantially similar” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 90%, preferably atleast 95% sequence identity to the reference sequence over a comparisonwindow of 10-20 amino acids. Percentage of sequence identity isdetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The phrase “disulfide bond” or “cysteine-cysteine disulfide bond” refersto a covalent interaction between two cysteines in which the sulfuratoms of the cysteines are oxidized to form a disulfide bond. Theaverage bond energy of a disulfide bond is about 60 kcal/mol compared to1-2 kcal/mol for a hydrogen bond. In the context of this invention, thecysteines which form the disulfide bond are within the framework regionsof the single chain antibody and serve to stabilize the conformation ofthe antibody.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule. In thecontext of the present invention, the terms include reference to joiningan antibody moiety to an effector molecule (EM). The linkage can beeither by chemical or recombinant means. Chemical means refers to areaction between the antibody moiety and the effector molecule such thatthere is a covalent bond formed between the two molecules to form onemolecule.

As used herein, “recombinant” includes reference to a protein producedusing cells that do not have, in their native state, an endogenous copyof the DNA able to express the protein. The cells produce therecombinant protein because they have been genetically altered by theintroduction of the appropriate isolated nucleic acid sequence. The termalso includes reference to a cell, or nucleic acid, or vector, that hasbeen modified by the introduction of a heterologous nucleic acid or thealteration of a native nucleic acid to a form not native to that cell,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell, express mutants of genes that arefound within the native form, or express native genes that are otherwiseabnormally expressed, underexpressed or not expressed at all.

As used herein, “nucleic acid” or “nucleic acid sequence” includesreference to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence includesthe complementary sequence thereof as well as conservative variants,i.e., nucleic acids present in wobble positions of codons and variantsthat, when translated into a protein, result in a conservativesubstitution of an amino acid.

As used herein, “encoding” with respect to a specified nucleic acid,includes reference to nucleic acids which comprise the information fortranslation into the specified protein. The information is specified bythe use of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolum (Proc. Nat'l Acad.Sci. USA 82:2306-2309 (1985), or the ciliate Macronucleus, may be usedwhen the nucleic acid is expressed in using the translational machineryof these organisms.

The phrase “fusing in frame” refers to joining two or more nucleic acidsequences which encode polypeptides so that the joined nucleic acidsequence translates into a single chain protein which comprises theoriginal polypeptide chains.

As used herein, “expressed” includes reference to translation of anucleic acid into a protein. Proteins may be expressed and remainintracellular, become a component of the cell surface membrane or besecreted into the extracellular matrix or medium.

By “host cell” is meant a cell which can support the replication orexpression of the expression vector. Host cells may be prokaryotic cellssuch as E. coli, or eukaryotic cells such as yeast, insect, amphibian,or mammalian cells.

The phrase “phage display library” refers to a population ofbacteriophage, each of which contains a foreign cDNA recombinantly fusedin frame to a surface protein. The phage display the foreign proteinencoded by the cDNA on its surface. After replication in a bacterialhost, typically E. coli, the phage which contain the foreign cDNA ofinterest are selected by the expression of the foreign protein on thephage surface.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, more preferably 65%, even more preferably 70%,still more preferably 75%, even more preferably 80%, and most preferably90-95% nucleotide or amino acid residue identity, when compared andaligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection.Preferably, the substantial identity exists over a region of thesequences that is at least about 50 residues in length, more preferablyover a region of at least about 100 residues, and most preferably thesequences are substantially identical over at least about 150 residues.In a most preferred embodiment, the sequences are substantiallyidentical over the entire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. 1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

The term “in vivo” includes reference to inside the body of the organismfrom which the cell was obtained. “Ex vivo” and “in vitro” means outsidethe body of the organism from which the cell was obtained.

The phrase “malignant cell” or “malignancy” refers to tumors or tumorcells that are invasive and/or able to undergo metastasis, i.e., acancerous cell.

As used herein, “mammalian cells” includes reference to cells derivedfrom mammals including humans, rats, mice, guinea pigs, chimpanzees, ormacaques. The cells may be cultured in vivo or in vitro.

The term “selectively reactive” refers, with respect to an antigen, thepreferential association of an antibody, in whole or part, with a cellor tissue bearing that antigen and not to cells or tissues lacking thatantigen. It is, of course, recognized that a certain degree ofnon-specific interaction may occur between a molecule and a non-targetcell or tissue. Nevertheless, selective reactivity, may be distinguishedas mediated through specific recognition of the antigen. Althoughselectively reactive antibodies bind antigen, they may do so with lowaffinity. On the other hand, specific binding results in a much strongerassociation between the antibody and cells bearing the antigen thanbetween the bound antibody and cells lacking the antigen. Specificbinding typically results in greater than 2-fold, preferably greaterthan 5-fold, more preferably greater than 10-fold and most preferablygreater than 100-fold increase in amount of bound antibody (per unittime) to a cell or tissue bearing CD22 as compared to a cell or tissuelacking CD22. Specific binding to a protein under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. A variety of immunoassay formats are appropriate forselecting antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See Harlow & Lane, ANTIBODIES, A LABORATORY MANUAL, Cold SpringHarbor Publications, New York (1988), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

The term “immunologically reactive conditions” includes reference toconditions which allow an antibody generated to a particular epitope tobind to that epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. Preferably, the immunologically reactive conditions employedin the methods of the present invention are “physiological conditions”which include reference to conditions (e.g., temperature, osmolarity,pH) that are typical inside a living mammal or a mammalian cell. Whileit is recognized that some organs are subject to extreme conditions, theintra-organismal and intracellular environment normally lies around pH 7(i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), containswater as the predominant solvent, and exists at a temperature above 0°C. and below 50° C. Osmolarity is within the range that is supportive ofcell viability and proliferation.

Numbering of Amino Acid Residues in the RFB4 Heavy and Light Chains

The positions of amino acid residues in an antibody heavy chain or lightchain are conveniently referred to in the art by standard numbering asset forth in Kabat, E., et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICALINTEREST, U.S. Government Printing Office, NIH Publication No. 91-3242(1991). See also, Johnson, G. and Wu, T., Nuc. Acids Res. 29:205-206(2001). The Kabat et al. database is typically referred to in the art aseither “Kabat” or “Kabat and Wu”. It is now maintained on-line athttp://immuno.bme.nwu.edu/. The heavy and light chains of RFB4 have beencloned. See, Mansfield et al., Blood 90:2020-2026 (1997). The amino acidsequences of the RFB4 V_(L) and V_(H) chains and a list of the Kabatnumbering of the position of each amino acid residue are set forth inthe Kabat database under Entry Numbers 038145 and 038146, respectively.FIG. 2 shows the comparison of the numbering of the amino acids of theRFB4 V_(L) chain to the corresponding Kabat positions as set forth inKabat Entry 038145; FIG. 3 shows the same comparison for the amino acidsof the RFB4 Via chain, as set forth in Kabat Entry 038146.

Binding of Antibodies and Immunoassays

A. Binding Affinity of Antibodies

The antibodies of this invention bind to their target antigens with anaffinity better than that of the parental RFB4 antibody. The antibodiesare anti-CD22 antibodies which bind to an extracellular epitope of CD22.Binding affinity for a target antigen is typically measured ordetermined by standard antibody-antigen assays, such as competitiveassays, saturation assays, or immunoassays such as ELISA or RIA.

Such assays can be used to determine the dissociation constant of theantibody. The phrase “dissociation constant” refers to the affinity ofan antibody for an antigen. Specificity of binding between an antibodyand an antigen exists if the dissociation constant (K_(D)=1/K, where Kis the affinity constant) of the antibody is <1 μM, preferably <100 nM,and most preferably <0.1 nM. Antibody molecules will typically have aK_(D) in the lower ranges. K_(D)=[Ab-Ag]/[Ab][Ag] where [Ab] is theconcentration at equilibrium of the antibody, [Ag] is the concentrationat equilibrium of the antigen and [Ab-Ag] is the concentration atequilibrium of the antibody-antigen complex. Typically, the bindinginteractions between antigen and antibody include reversible noncovalentassociations such as electrostatic attraction, Van der Waals forces andhydrogen bonds. This method of defining binding specificity applies tosingle heavy and/or light chains, CDRs, fusion proteins or fragments ofheavy and/or light chains, that are specific for CD22 if they bind CD22alone or in combination.

B. Immunoassays

The antibodies can be detected and/or quantified using any of a numberof well recognized immunological binding assays (see, e.g., U.S. Pat.Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review ofthe general immunoassays, see also METHODS IN CELL BIOLOGY, VOL. 37,Asai, ed. Academic Press, Inc. New York (1993); BASIC AND CLINICALIMMUNOLOGY 7TH EDITION, Stites & Terr, eds. (1991). Immunologicalbinding assays (or immunoassays) typically utilize a ligand (e.g., CD22)to specifically bind to and often immobilize an antibody. The antibodiesemployed in immunoassays of the present invention are discussed ingreater detail supra.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the ligand and the antibody. Thelabeling agent may itself be one of the moieties comprising theantibody/analyte complex, i.e., the anti-CD22 antibody. Alternatively,the labeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/CD22 protein complex.

In one aspect, a competitive assay is contemplated wherein the labelingagent is a second anti-CD22 antibody bearing a label. The two antibodiesthen compete for binding to the immobilized CD22. Alternatively, in anon-competitive format, the CD22 antibody lacks a label, but a secondantibody specific to antibodies of the species from which the anti-CD22antibody is derived, e.g., murine, and which binds the anti-CD22antibody, is labeled.

Other proteins capable of specifically binding immunoglobulin constantregions, such as Protein A or Protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al., J. Immunol. 111:1401-1406 (1973); andAkerstrom, et al., J. Immunol. 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antibody, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

While the details of the immunoassays of the present invention may varywith the particular format employed, the method of detecting anti-CD22antibodies in a sample containing the antibodies generally comprises thesteps of contacting the sample with an antibody which specificallyreacts, under immunologically reactive conditions, to the CD22/antibodycomplex.

Production of Immunoconjugates

Immunoconjugates include, but are not limited to, molecules in whichthere is a covalent linkage of a therapeutic agent to an antibody. Atherapeutic agent is an agent with a particular biological activitydirected against a particular target molecule or a cell bearing a targetmolecule. One of skill in the art will appreciate that therapeuticagents may include various drugs such as vinblastine, daunomycin and thelike, cytotoxins such as native or modified Pseudomonas exotoxin orDiphtheria toxin, encapsulating agents, (e.g., liposomes) whichthemselves contain pharmacological compositions, radioactive agents suchas ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and other labels, target moieties andligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell and the biological effect is desired to evoke.Thus, for example, the therapeutic agent may be a cytotoxin which isused to bring about the death of a particular target cell. Conversely,where it is merely desired to invoke a non-lethal biological response,the therapeutic agent may be conjugated to a non-lethal pharmacologicalagent or a liposome containing a non-lethal pharmacological agent.

With the therapeutic agents and antibodies herein provided, one of skillcan readily construct a variety of clones containing functionallyequivalent nucleic acids, such as nucleic acids which differ in sequencebut which encode the same EM or antibody sequence. Thus, the presentinvention provides nucleic acids encoding antibodies and conjugates andfusion proteins thereof.

A. Recombinant Methods

The nucleic acid sequences of the present invention can be prepared byany suitable method including, for example, cloning of appropriatesequences or by direct chemical synthesis by methods such as thephosphotriester method of Narang, el al., Meth. Enzymol. 68:90-99(1979); the phosphodiester method of Brown, et al., Meth. Enzymol.68:109-151 (1979); the diethylphosphoramidite method of Beaucage, etal., Tetra. Lett. 22:1859-1862 (1981); the solid phase phosphoramiditetriester method described by Beaucage & Caruthers, Tetra. Letts.22(20):1859-1862 (1981), e.g., using an automated synthesizer asdescribed in, for example, Needham-VanDevanter, et al. Nucl. Acids Res.12:6159-6168 (1984); and, the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

In a preferred embodiment, the nucleic acid sequences of this inventionare prepared by cloning techniques. Examples of appropriate cloning andsequencing techniques, and instructions sufficient to direct persons ofskill through many cloning exercises are found in Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold SpringHarbor Laboratory (1989)), Berger and Kimmel (eds.), GUIDE TO MOLECULARCLONING TECHNIQUES, Academic Press, Inc., San Diego Calif. (1987)), orAusubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing and Wiley-Interscience, NY (1987). Product information frommanufacturers of biological reagents and experimental equipment alsoprovide useful information. Such manufacturers include the SIGMAchemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Nucleic acids encoding native EM or anti-CD22 antibodies can be modifiedto form the EM, antibodies, or immunoconjugates of the presentinvention. Modification by site-directed mutagenesis is well known inthe art. Nucleic acids encoding EM or anti-CD22 antibodies can beamplified by in vitro methods. Amplification methods include thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (3SR). A wide variety of cloning methods,host cells, and in vitro amplification methodologies are well known topersons of skill.

In a preferred embodiment, immunoconjugates are prepared by insertingthe cDNA which encodes an anti-CD22 scFv antibody into a vector whichcomprises the cDNA encoding the EM. The insertion is made so that thescFv and the EM are read in frame, that is in one continuous polypeptidewhich contains a functional Fv region and a functional EM region. In aparticularly preferred embodiment, cDNA encoding a diphtheria toxinfragment is ligated to a scFv so that the toxin is located at thecarboxyl terminus of the scFv. In more preferred embodiments, cDNAencoding PE is ligated to a scFv so that the toxin is located at theamino terminus of the scFv.

Once the nucleic acids encoding an EM, anti-CD22 antibody, or animmunoconjugate of the present invention are isolated and cloned, onemay express the desired protein in a recombinantly engineered cell suchas bacteria, plant, yeast, insect and mammalian cells. It is expectedthat those of skill in the art are knowledgeable in the numerousexpression systems available for expression of proteins including E.coli, other bacterial hosts, yeast, and various higher eucaryotic cellssuch as the COS, CHO, HeLa and myeloma cell lines. No attempt todescribe in detail the various methods known for the expression ofproteins in prokaryotes or eukaryotes will be made. In brief, theexpression of natural or synthetic nucleic acids encoding the isolatedproteins of the invention will typically be achieved by operably linkingthe DNA or cDNA to a promoter (which is either constitutive orinducible), followed by incorporation into an expression cassette. Thecassettes can be suitable for replication and integration in eitherprokaryotes or eukaryotes. Typical expression cassettes containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the DNA encodingthe protein. To obtain high level expression of a cloned gene, it isdesirable to construct expression cassettes which contain, at theminimum, a strong promoter to direct transcription, a ribosome bindingsite for translational initiation, and a transcription/translationterminator. For E. coli this includes a promoter such as the T7, trp,lac, or lambda promoters, a ribosome binding site and preferably atranscription termination signal. For eukaryotic cells, the controlsequences can include a promoter and preferably an enhancer derived fromimmunoglobulin genes, SV40, cytomegaloviius, and a polyadenylationsequence, and may include splice donor and acceptor sequences. Thecassettes of the invention can be transferred into the chosen host cellby well-known methods such as calcium chloride transformation orelectroporation for E. coli and calcium phosphate treatment,electroporation or lipofection for mammalian cells. Cells transformed bythe cassettes can be selected by resistance to antibiotics conferred bygenes contained in the cassettes, such as the amp, gpt, neo and hyggenes.

One of skill would recognize that modifications can be made to a nucleicacid encoding a polypeptide of the present invention (i.e., anti-CD22antibody, PE, or an immunoconjugate formed from their combination)without diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, terminationcodons, a methionine added at the amino terminus to provide aninitiation, site, additional amino acids placed on either terminus tocreate conveniently located restriction sites, or additional amino acids(such as poly His) to aid in purification steps.

In addition to recombinant methods, the immunoconjugates, EM, andantibodies of the present invention can also be constructed in whole orin part using standard peptide synthesis. Solid phase synthesis of thepolypeptides of the present invention of less than about 50 amino acidsin length may be accomplished by attaching the C-terminal amino acid ofthe sequence to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, THE PEPTIDES: ANALYSIS,SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS, PARTA. pp. 3-284; Merrifield, et al. J. Am. Chem. Soc. 85:2149-2156 (1963),and Stewart, et al., SOLID PHASE PEPTIDE SYNTHESIS, 2ND ED., PierceChem. Co., Rockford, Ill. (1984). Proteins of greater length may besynthesized by condensation of the amino and carboxyl termini of shorterfragments. Methods of forming peptide bonds by activation of a carboxylterminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

B. Purification

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules of the present invention can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N.Y.(1982)). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, if to be used therapeutically, the polypeptidesshould be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies of this invention. See, Buchner, etal.,Anal. Biochem. 205:263-270 (1992); Pluckthun, Biotechnology 9:545(1991); Huse, et al., Science 246:1275 (1989) and Ward, et al., Nature341:544 (1989), all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well-known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena, et al., Biochemistry 9: 5015-5021 (1970),incorporated by reference herein, and especially as described byBuchner, et al., supra.

Renaturation is typically accomplished by dilution (e.g., 100-fold) ofthe denatured and reduced protein into refolding buffer. An exemplarybuffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. A preferred yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. It isdesirable to add excess oxidized glutathione or other oxidizing lowmolecular weight compounds to the refolding solution after theredox-shuffling is completed.

Pseudomonas Exotoxin and Other Toxins

Toxins can be employed with antibodies of the present invention to yieldimmunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxinand subunits thereof, as well as botulinum toxins A through F. Thesetoxins are readily available from commercial sources (e.g., SigmaChemical Company, St. Louis, Mo.). Diphtheria toxin is isolated fromCorynebaclerium diphtheriae. Ricin is the lectin RCA60 from Ricinuscommunis (Castor bean). The term also references toxic variants thereof.For example, see, U.S. Pat. Nos. 5,079,163 and 4,689,401. Ricinuscommunis agglutinin (RCA) occurs in two forms designated RCA₆₀ andRCA₁₂₀ according to their molecular weights of approximately 65 and 120kD, respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta266:543 (1972)). The A chain is responsible for inactivating proteinsynthesis and killing cells. The B chain binds ricin to cell-surfacegalactose residues and facilitates transport of the A chain into thecytosol (Olsnes, et al., Nature 249:627-631 (1974) and U.S. Pat. No.3,060,165).

Abrin includes toxic lectins from Abrus precalorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 kD and are composed of two disulfide-linked polypeptide chainsA and B. The A chain inhibits protein synthesis; the B-chain (abrin-b)binds to D-galactose residues (see, Funatsu, et al., Agr. Biol. Chem.52:1095 (1988); and Olsnes, Methods Enzymol. 50:330-335 (1978)).

In preferred embodiments of the present invention, the toxin isPseudomonas exotoxin (PE). The term “Pseudomonas exotoxin” as usedherein refers to a full-length native (naturally occurring) PE or a PEthat has been modified. Such modifications may include, but are notlimited to, elimination of domain Ia, various amino acid deletions indomains lb, II and III, single amino acid substitutions and the additionof one or more sequences at the carboxyl terminus such as KDEL (SEQ IDNO:5) and REDL (SEQ ID NO:6). See Siegall, el al., J. Biol. Chem.264:14256-14261 (1989). In a preferred embodiment, the cytotoxicfragment of PE retains at least 50%, preferably 75%, more preferably atleast 90%, and most preferably 95% of the cytotoxicity of native PE. Ina most preferred embodiment, the cytotoxic fragment is more toxic thannative PE.

Native Pseudomonas exotoxin A (PE) is an extremely active monomericprotein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa,which inhibits protein synthesis in eukaryotic cells. The native PEsequence is provided in commonly assigned U.S. Pat. No. 5,602,095,incorporated herein by reference. The method of action is inactivationof the ADP-ribosylation of elongation factor 2 (EF-2). The exotoxincontains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2. The function of domain lb (aminoacids 365-399) remains undefined, although a large part of it, aminoacids 365-380, can be deleted without loss of cytotoxicity. See Siegall,et al., (1989), supra.

PE employed in the present invention include the native sequence,cytotoxic fragments of the native sequence, and conservatively modifiedvariants of native PE and its cytotoxic fragments. Cytotoxic fragmentsof PE include those which are cytotoxic with or without subsequentproteolytic or other processing in the target cell (e.g., as a proteinor pre-protein). Cytotoxic fragments of PE include PE40, PE38, and PE35.

In preferred embodiments, the PE has been modified to reduce oreliminate non-specific cell binding, frequently by deleting domain Ia.as taught in U.S. Pat. No. 4,892,827, although this can also beachieved, for example, by mutating certain residues of domain Ia. U.S.Pat. No. 5,512,658, for instance, discloses that a mutated PE in whichDomain Ia is present but in which the basic residues of domain Ia atpositions 57, 246, 247, and 249 are replaced with acidic residues(glutamic acid, or “E”)) exhibits greatly diminished non-specificcytotoxicity. This mutant form of PE is sometimes referred to as PE4E.

PE40 is a truncated derivative of PE as previously described in the art.See, Pai, et al., Proc. Nat'l Acad. Sci. USA 88:3358-62 (1991); andKondo, et al., J. Biol. Chem. 263:9470-9475 (1988). PE35 is a 35 kDcarboxyl-terminal fragment of PE in which amino acid residues 1-279 havedeleted and the molecule commences with a met at position 280 followedby amino acids 281-364 and 381-613 of native PE. PE35 and PE40 aredisclosed, for example, in U.S. Pat. Nos. 5,602,095 and 4,892,827.

In some preferred embodiments, the cytotoxic fragment PE38 is employed.PE38 is a truncated PE pro-protein composed of amino acids 253-364 and381-613 which is activated to its cytotoxic form upon processing withina cell (see e.g., U.S. Pat. No. 5,608,039, and Pastan et al., Biochim.Biophys. Acta 1333:C₁-C₆ (1997)).

As noted above, some or all of domain lb may be deleted, and theremaining portions joined by a linker or directly by a peptide bond.Some of the amino portion of domain II may be deleted. And, theC-terminal end may contain the native sequence of residues 609-613(REDLK (SEQ ID NO:7)), or may contain a variation found to maintain theability of the construct to translocate into the cytosol, such as REDL(SEQ ID NO:6) or KDEL (SEQ ID NO:5), and repeats of these sequences.See, e.g., U.S. Pat. Nos. 5,854,044; 5,821,238; and 5,602,095 and WO99/51643. While in preferred embodiments, the PE is PE4E, PE40, or PE38,any form of PE in which non-specific cytotoxicity has been eliminated orreduced to levels in which significant toxicity to non-targeted cellsdoes not occur can be used in the immunotoxins of the present inventionso long as it remains capable of translocation and EF-2 ribosylation ina targeted cell.

A. Conservatively Modified Variants of PE

Conservatively modified variants of PE or cytotoxic fragments thereofhave at least 80% sequence similarity, preferably at least 85% sequencesimilarity, more preferably at least 90% sequence similarity, and mostpreferably at least 95% sequence similarity at the amino acid level,with the PE of interest, such as PE38.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidsequences which encode identical or essentially identical amino acidsequences, or if the nucleic acid does not encode an amino acidsequence, to essentially identical nucleic acid sequences. Because ofthe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given polypeptide. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine) canbe modified to yield a functionally identical molecule. Accordingly,each silent variation of a nucleic acid which encodes a polypeptide isimplicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.

B. Assaying for Cytotoxicity of PE

Pseudomonas exotoxins employed in the invention can be assayed for thedesired level of cytotoxicity by assays well known to those of skill inthe art. Thus, cytotoxic fragments of PE and conservatively modifiedvariants of such fragments can be readily assayed for cytotoxicity. Alarge number of candidate PE molecules can be assayed simultaneously forcytotoxicity by methods well known in the art. For example, subgroups ofthe candidate molecules can be assayed for cytotoxicity. Positivelyreacting subgroups of the candidate molecules can be continuallysubdivided and reassayed until the desired cytotoxic fragment(s) isidentified. Such methods allow rapid screening of large numbers ofcytotoxic fragments or conservative variants of PE.

C. Other Therapeutic Moieties

Antibodies of the present invention can also be used to target anynumber of different diagnostic or therapeutic compounds to cellsexpressing CD22 on their surface. Thus, an antibody of the presentinvention, such as an anti-CD22 scFv, may be attached directly or via alinker to a drug that is to be delivered directly to cells bearing CD22.Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides.

Alternatively, the molecule linked to an anti-CD22 antibody may be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g. anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735;and Connor, et al., Pharm. Ther. 28:341-365 (1985).

D. Detectable Labels

Antibodies of the present invention may optionally be covalently ornon-covalently linked to a detectable label. Detectable labels suitablefor such use include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. DYNABEADS), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

E. Conjugation to the Antibody

In a non-recombinant embodiment of the invention, effector molecules,e.g., therapeutic, diagnostic, or detection moieties, are linked to theanti-CD22 antibodies of the present invention using any number of meansknown to those of skill in the art. Both covalent and noncovalentattachment means may be used with anti-CD22 antibodies of the presentinvention.

The procedure for attaching an effector molecule to an antibody willvary according to the chemical structure of the EM. Polypeptidestypically contain a variety of functional groups; e.g., carboxylic acid(COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on an antibodyto result in the binding of the effector molecule.

Alternatively, the antibody is derivatized to expose or to attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford Ill.

A “linker”, as used herein, is a molecule that is used to join theantibody to the effector molecule. The linker is capable of formingcovalent bonds to both the antibody and to the effector molecule.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (e.g.,through a disulfide linkage to cysteine). However, in a preferredembodiment, the linkers will be joined to the alpha carbon amino andcarboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages which are cleavable in the vicinity of the target site.Cleavage of the linker to release the effector molecule from theantibody may be prompted by enzymatic activity or conditions to whichthe immunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

Pharmaceutical Compositions and Administration

The antibody and/or immunoconjugate compositions of this invention(i.e., PE linked to an anti-CD22 antibody of the invention) areparticularly useful for parenteral administration, such as intravenousadministration or administration into a body cavity.

The compositions for administration will commonly comprise a solution ofthe antibody and/or immunoconjugate dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of fusion protein in these formulations can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs.

Thus, a typical pharmaceutical immunotoxin composition of the presentinvention for intravenous administration would be about 0.1 to 10 mg perpatient per day. Dosages from 0.1 up to about 100 mg per patient per daymay be used. Actual methods for preparing administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as REMINGTON'S PHARMACEUTICALSCIENCE, 19TH ED., Mack Publishing Company, Easton, Pa. (1995).

The compositions of the present invention can be administered fortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. An effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the proteins of this invention to effectively treat thepatient. Preferably, the dosage is administered once but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the patient.

Controlled release parenteral formulations of the immunoconjugatecompositions of the present invention can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS:FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μm so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly. See,e.g., Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed.,Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice &Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., MarcelDekker, Inc. New York, N.Y., pp. 315-339, (1992) both of which areincorporated herein by reference.

Polymers can be used for ion-controlled release of immunoconjugatecompositions of the present invention. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)).For example, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston, etal., Pharm. Res. 9:425-434 (1992); and Pec, et al., J. Parent. Sci.Tech. 44 (2): 58-65 (1990)). Alternatively, hydroxyapatite has been usedas a microcarrier for controlled release of proteins (Ijntema, etal.,Int. J. Pharm. 112:215-224(1994)). In yet another aspect, liposomes areused for controlled release as well as drug targeting of thelipid-capsulated drug (Betageri, et al., LIPOSOME DRUG DELIVERY SYSTEMS,Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerousadditional systems for controlled delivery of therapeutic proteins areknown. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871,4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670;5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961;5,254,342 and 5,534,496, each of which is incorporated herein byreference.

Among various uses of the immunotoxins of the present invention areincluded a variety of disease conditions caused by specific human cellsthat may be eliminated by the toxic action of the fusion protein. Onepreferred application for the immunotoxins of the invention is thetreatment of malignant cells expressing CD22. Exemplary malignant cellsinclude those of chronic lymphocytic leukemia and hairy cell leukemia.

Diagnostic Kits and In Vitro Uses

In another embodiment, this invention provides for kits for thedetection of CD22 or an immunoreactive fragment thereof, (i.e.,collectively, a “CD22 protein”) in a biological sample. A “biologicalsample” as used herein is a sample of biological tissue or fluid thatcontains CD22. Such samples include, but are not limited to, tissue frombiopsy, blood, and blood cells (e.g., white cells). Preferably, thecells are lymphocytes. Biological samples also include sections oftissues, such as frozen sections taken for histological purposes. Abiological sample is typically obtained from a multicellular eukaryote,preferably a mammal such as rat, mouse, cow, dog, guinea pig, or rabbit,and more preferably a primate, such as a macaque, chimpanzee, or human.Most preferably, the sample is from a human.

Kits will typically comprise an anti-CD22 antibody of the presentinvention. In some embodiments, the anti-CD22 antibody will be ananti-CD22 Fv fragment, such as a scFv or dsFv fragment.

In addition the kits will typically include instructional materialsdisclosing means of use of an antibody of the present invention (e.g.for detection of mesothelial cells in a sample). The kits may alsoinclude additional components to facilitate the particular applicationfor which the kit is designed. Thus, for example, the kit mayadditionally contain means of detecting the label (e.g. enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a sheep anti-mouse-HRP, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

In one embodiment of the present invention, the diagnostic kit comprisesan immunoassay. As described above, although the details of theimmunoassays of the present invention may vary with the particularformat employed, the method of detecting CD22 in a biological samplegenerally comprises the steps of contacting the biological sample withan antibody of the present invention which specifically reacts, underimmunologically reactive conditions, to CD22. The antibody is allowed tobind to CD22 under immunologically reactive conditions, and the presenceof the bound antibody is detected directly or indirectly.

Due to the increased affinity of the antibodies of the invention, theantibodies will be especially useful as diagnostic agents and in invitro assays to detect the presence of CD22 in biological samples. Forexample, the antibodies taught herein can be used as the targetingmoieties of immunoconjugates in immunohistochemical assays to determinewhether a sample contains cells expressing CD22. Detection of CD22 inlymphocytes would indicate either that the patient has a cancercharacterized by the presence of CD22-expressing cells, or that atreatment for such a cancer has not yet been successful at eradicatingthe cancer.

In another set of uses for the invention, immunotoxins targeted byantibodies of the invention can be used to purge targeted cells from apopulation of cells in a culture. Thus, for example, cells cultured froma patient having a cancer expressing CD22 can be purged of cancer cellsby contacting the culture with immunotoxins which use the antibodies ofthe invention as a targeting moiety.

EXAMPLES Example 1

The experiments reported in this example demonstrate the creation anduse of of phage display libraries to select RFB4-Fvs that bind the CD22antigen of Daudi cells with increased affinity over the wild typeRBF4-Fv.

The CDR3 of the variable heavy chain (V_(H)) of RFB4 (Fv) was mutated inan attempt to create Fvs with increased antigen binding affinity. Thewild type amino acid sequence of V_(H)CDR3 of RFB4 (Fv) contains 14amino acids, as shown below in Table 1.

TABLE 1 DNA and amino acid sequences of CDR3 heavy chain RFB495  96  97  98  99 100 100A 100B 100C 100D 100E 100F 101 102 H   S   G   Y   G   S  S    Y    G    V    L    F    A   Y CAT AGTGGC TAC GGT AGT AGC TAC  GGG  GTT  TTG  TTT  GCT TAC                    _(——————)                         _(—————)

The mutational hot spots in V_(H) CDR3 of RFB4 are underlined in theTable. A selected subgroup of the hotspots was targeted for mutagenesis:G99 (GGT), S100 (AGT), S100A (AGC), and Y100B (TAC) were randomlymutated and a library of 1.6×10⁵ clones was produced. The residuesmutated are shown in bold in the Table. The numbering of the residuesfollows the Kabat format.

To create a template for the construction of the library, PCR was usedto amplify RFB4-Fv from the plasmid pEM10 [RFB4 (scFv)-PE38 KDEL]. Thefollowing oligomers, which introduced SfiI and NotI restriction sitesinto the PCR product, were used for this amplfication:

SEQ ID NO. 8: 5′TTCTATGCGGCCCAGCCGCCATGGCCGAAGTGCAGCTGGTGGAGTCT- 3′ SEQID NO. 9: 5′CGGCACCGGCGCACCTGCGGCCGCCCGTTGATTTCCAGCTTGGTGCC- 3′.

The resulting PCR product was digested with SfiI and NotI and insertedinto the vector, pCANTAB5E (Pharmacia). The resulting phagemid,pCANTAB5E-RFB4, was then modified by inserting a stop codon (TAA) atposition 99 (GGT) using site directed mutagenesis (Quick Change™site-directed mutagenesis Kit, Stratagene). The final phagemid product,pCANTAB5E-RFB4-1, was used as the template for the introduction of thefour amino acid randomizations in the V_(H)CDR3 region.

DNA oligomers twelve nucleotides in length, were designed to generate alibrary randomizing the four chosen consecutive amino acids. Degenerateoligomers with the sequence NNS were used, where N is any of the fournucleotides, and where S is C or G. The following oligonucleotides wereused to create the library:

SEQ ID NO. 10: 5′-CAACGTGAAAAAATTAATTATTCGC SEQ ID NO. 11:5′-AGCAAACAAACCCCSNNSNNSNNSNNGTAGCCACTATGTCT SEQ ID NO. 12:5′-GCTAAACAACTTTCAACAGTCTATGCGGGCAC

The library was constructed by employing two sequential PCR reactions.In the first PCR, 50 pg of the phagemid template, pCANTAB5E-RFB4-1, wascombined with 20 pmol each of DNA oligomers SEQ ID No. 3 and SEQ ID No.4, mixed with two Ready-To-Go PCR beads (Pharmacia) in a 50 μl volumeand cycled using the following profile: 1 cycle at 95° C. for 5 min,followed by 30 cycles at 94° C. for 1 min, 55° C. for 1 min, and 72° C.for 1 min. The PCR reaction generated a 402 base pair product containingthe mutations. The product was purified using a Qiagen Quick Spin columnand quantitated by visualization on a 1% agarose gel. The purifiedproduct was used as primers in a second PCR.

In the second PCR, 2 pmol of product from the first PCR reaction wascombined with 20 pmol of the DNA oligomer SEQ ID No. 5, 50 pg ofphagemid pCANTAB5E-RFB4-1 template, mixed with two PCR beads in 50 μlvolume, and cycled using the profile described above. The reactiongenerated an 884 base pair insert library. The 884 bp PCR product wasdigested with SfiI and NotI, purified using a Qiaquick column (Qiagen),and 150 ng was ligated into 250 ng of the phage display vectorpCANTAB5E, and desalted. Forty nanograms of the ligation were used totransform E. coli TGI. Ten transformations were performed to give alibrary containing 8×10⁵ clones. The phage library was rescued from thetransformed bacteria, as previously described (Beers, R. et al., Clin.Cancer Res., 6:2835-2843 (2000)), titered and stored at 4° C. To testwhether the library was properly randomized, 16 clones were sequencedthrough the mutated region. Each clone had a different DNA sequence,thus indicating that a well randomized library for the selection ofscFvs with high affinity for binding of the CD22 antigen had beencreated.

Phage were rescued from the library and panned on Daudi cells, whichhave 1×10⁵ CD22 binding sites (Shen, G. L. et al., Int. J. Cancer,42:792-797 (1988)). Cells (2×10⁷) were pelleted, resuspended in 10 ml ofcold blocking buffer (DPBS+0.5% BSA+5 mM EDTA) and rotated slowly for 90min at 4° C. The cells were then pelleted and resuspended in 1 ml ofcold blocking buffer. Phage 1×10¹²) from the library were added to thecell suspension and the mixture was rotated slowly at 4° C. for 90 min.The cells were washed five times with 10 ml of cold blocking buffer.Bound phage were eluted by resuspending the washed cells in 1.5 ml ofice cold 50 mM HCL and incubating on ice for 10 min. Daudi cells werepelleted and the eluted phage were transferred to a tube containing 200μl of 1 M TRIS pH 8. The eluted phage were titered to determine thenumber of phage captured. 1.5 ml of the eluted phage were reinfectedinto E. coli and amplified for use in the next round of panning. Toavoid possible loss of high affinity Fvs, panning was limited to tworounds only (Beers, R. et al., Clin. Cancer Res., 6:2835-2843 (2000)). A60-fold enrichment was achieved between round 1 and round 2.

After the second round of panning, phage stocks were prepared fromtwenty-four individual clones and tested for their ability to bind toDaudi cells by flow cytometry. Single colonies of E. coli TG1 containingphagemids selected in the 2^(nd) round of panning were grown toOD₆₀₀=0.3 in 15 ml of 2× YT medium supplemented with 2% glucose andampicillin (100 μg/ml). M13KO7 helper phage (10¹⁰ PFU) was added to thesuspension and cells were incubated for 1 h at 37° C. Followingincubation, the mixture was centrifuged, resuspended in 30 ml of 2xYTplus ampicillin (100 μg/ml) and kanamycin (50 μg/ml) and grown 16 h at37° C. Following growth, the cultures were pelleted and phage wereprecipitated from the supernatant with PEG/NaCl. After centrifugation,the phage pellets were each resuspended in 1 ml of NTE (100 mM NaCl, 10mM Tris [pH 7.5] and 1 mM EDTA) and titered.

To determine the binding properties of the 24 phage stocks acquiredafter the second round of panning, phage were mixed with Daudi cells,reacted with a primary anti-M13 antibody followed by reaction withsecondary FITC-labeled antibody and finally, fluorescence was measuredby flow cytometry.

5×10⁵ Daudi cells were incubated with 8×10⁸ phage at room temperaturefor 60 min, cells were washed two times with blocking buffer (DPBS+0.5%BSA+5 mM EDTA) and 5 μg of mouse anti-M13 antibody (Amersham) was addedto each sample. The mixture was incubated at room temperature for 20min, then washed two times with blocking buffer. A goat-anti-mouse-FITClabeled antibody (Jackson ImmunoResearch) was added and cells wereincubated for 20 min at room temperature. Cells were washed two timesand analysis was performed in a FACSort flow cytometer (BectonDickinson). Data were acquired using Cell Quest software. For thecompetition experiment, 5×10⁶ cells were incubated with 8×10¹⁰ wild-typeRFB4 single chain Fv (scFv) phage and with 63 μg of RFB4 immunotoxin(100-fold excess). The sample was processed as for cells incubated withphage only.

Daudi cells incubated without phage generated only a background signalwhen analysed by flow cytometry. In contrast, the fluorescence intensitysignal generated by cells incubated with phage carrying an scFv bearingthe wild-type V_(H)CDR3 (GSSY) (SEQ ID NO:13) of RFB4 was significant.Cells that were co-incubated with phage carrying an scFv with thewild-type V_(H)CDR3 (GSSY) (SEQ ID NO:13) of RFB4 and the parental GSSY(SEQ ID NO:13) containing immunotoxin [RFB4 (Fv)-PE38], produced aflourescence signal similar to that of cells incubated without phage.Thus, phage that carry an scFv bearing the wild-type V_(H)CDR3 (GSSY)(SEQ ID NO:13) of RFB4 bind specifically to the CD22 antigen of Daudicells.

The fluorescence intensity generated by of Daudi cells that wereincubated with phage displaying an scFv with the wild-type V_(H)CDR3(GSSY) (SEQ ID NO:13) of RFB4 was compared to the fluorescence intensitysignal generated by incubation of Daudi cells with any one of threeother phage (A, B, and C) selected from the randomized library bypanning. The fluorescence intensity generated by incubation of of phageA and phage B with Daudi cells was greater than the fluorescenceintensity signal generated by Daudi cells incubated with phage carryingan scFv bearing the wild-type V_(H)CDR3 (GSSY) (SEQ ID NO:13) of RFB4.Thus, phage A and B carry scFvs that bind to the CD22 antigen of Daudicells better than phage carrying an scFv with the wild-type V_(H)CDR3(GSSY) (SEQ ID NO:13) of RFB4. Cells incubated with phage C had afluorescence intensity similar to that of cells incubated without phage,suggesting that this mutant did not bind the cells. Phage C wasclassified as a poor binder and was not analyzed further. Only two outof twenty four phage did not bind to the cells.

Twenty-two of the phage studied behaved like phage A and B. The Fvs ofthese 22 phage were sequenced using PE™ applied Biosystems Big DyeTerminator Cycle Sequencing Kit. The samples were run and analyzed on aPE Applied Biosystem Model 310 automated sequencer. The amino acidresidues of the region mutated in V_(H) CDR3 that were deduced from theDNA sequences are shown below in Table 2.

TABLE 2 Sequences of mutant phage obtained after panning 99 100 100A100B G S S Y (wild type) (SEQ ID NO: 13) G T H W (tested as an im- (SEQID NO: 14) munotoxin) G Y N W (tested as an im- (SEQ ID NO: 15)munotoxin) G T T W (tested as an im- (SEQ ID NO: 16) munotoxin) G S T Y(tested as an im- (SEQ ID NO: 17) munotoxin) G K N R (tested as an im-(SEQ ID NO: 18) munotoxin and found three times) G S T R (found twotimes) (SEQ ID NO: 19) G H T F (SEQ ID NO: 20) G N R Y (SEQ ID NO: 21) GT A Y (SEQ ID NO: 22) G T N Y (SEQ ID NO: 23) G L H Y (SEQ ID NO: 24) GF L Y (SEQ ID NO: 25) G S R Y (SEQ ID NO: 26) G R N Y (SEQ ID NO: 27) GV H R (SEQ ID NO: 28) G A L R (SEQ ID NO: 29) G V R A (SEQ ID NO: 30) GT A K (SEQ ID NO: 31) G R T S (SEQ ID NO: 32)The amino acid sequences of phage isolated after two rounds of panningis shown. The entire Fv of each phage was sequenced, however, only thesequence of the target region is shown.

The randomized library produced an abundance of mutations that resultedin an apparent increased binding affinity of the V_(H)CDR3 of RBF4 Fvfor the CD22 antigen of Daudi cells. Thus, a phage display library ofrandom mutants of the CDR3 region of the V_(H) of RFB4 was created andused to select RFB4 scFvs with improved binding affinity for the CD22antigen.

Example 2

The studies reported in this Example demonstrate that incorporation ofthe mutant scFvs selected in Example 1 into the structure of a chimericimmunotoxin molecule increases the cytotoxic activity of the chimericimmunotoxins toward cultured cells that display the CD22 antigen,thereby inhibiting growth of the cultures.

Immunotoxins from each of the three major 100B substitutions GTTW (SEQID NO:16), GYNW (SEQ ID NO:15), GTHW (SEQ ID NO:14), GSTY (SEQ IDNO:17), and GKNR (SEQ ID NO:18) were prepared. ScFvs from selectedphagemids were PCR amplified using primers that introduced Nde I andHindIII restrictions sites into the final PCR product. The products ofthe reaction were purified, digested with Nde I and HindIII and clonedinto a T7 expression vector in which the scFv was fused in frame to atruncated version of Pseudomonas exotoxin A (PE38) (Brinkmann, U., Mol.Med Today, 2:439-446 (1996)). The expression and purification of theresulting recombinant immunotoxins was performed as previously described(Beers, R. et al., Clin. Cancer Res., 6:2835-2843 (2000)).

Each immunotoxin was purified to over 95% homogeneity and eluted as amonomer using TSK gel filtration chromatography. The purifiedimmunotoxins were used in cytotoxicity assays on a panel of sixantigen-positive lymphoma cell lines.

Cytotoxicity on cell lines was measured by protein synthesis inhibitionassays. Cells were plated in 96-well plates at a concentration of 5×10⁴cells/well. Immunotoxins, prepared as described above, were seriallydiluted in phosphate-buffered saline (PBS)/0.2% human serum albumin(HSA) and 20 μl was added to each well. Plates were incubated for 20hours at 37° C. and then pulsed with 1 μCi/well ³H-leucine in 20 μLPBS/0.2% HSA for 2.5 hours at 37° C. Radiolabeled material was capturedon filtermats and counted in a Betaplate scintillation counter(Pharmacia, Gaithersburg, Md.). Triplicate sample values were averagedand inhibition of protein synthesis determined by calculating percentincorporation by comparison to control wells without added toxin. Theactivity of the molecule is defined by its IC₅₀, defined as the toxinconcentration that reduced incorporation of radioactivity by 50%compared with the cells that were not treated with the toxin. Table 3shows the IC₅₀ values from several experiments.

TABLE 3 Cytotoxic activity (IC₅₀) in ng/ml of selected RFB4-PE38 mutantson six different CD22-positive cell lines JD38 Ca46 raji Daudi namalwaRamos GSSY (WT) 2.3 ± 0.5  3.1 ± 0.2 5.1 ± 0.15 8.1 ± 2.3 10.6 ± 1.2 252 ± 3 (SEQ ID NO: 13) GTHW (SEQ 0.2 ± 0.09 1.4 ± 0.5   1 ± 0.14 1.7 ±0.1 2.8 ± 0.4  32 ± 3 ID NO: 14) GYNW 0.6 ± 0.1    0.8⁺ 0.6 ± 0.07 2.1 ±0.5 5.6 ± 3.3 N.D. (SEQ ID NO: 15) GTTW (SEQ 0.7 ± 0.03 1.7 ± 0.4 2.75 ±0.3  2.0 ± 1.2 5.3 ± 0.2 N.D. ID NO: 16) GSTY (SEQ   1.1⁺ 2⁺ 4.1⁺ 8.5 ±0.7    9.5⁺ N.D. ID NO: 17) GKNR (SEQ 5⁺ 6⁺ 10⁺   55 ± 7  25⁺ N.D. IDNO: 18) N.D.: not determined. ⁺Immunotoxin was tested once. All celllines were of Burkitt's lymphoma

All mutant immunotoxins except GKNR (SEQ ID NO:18) were more cytotoxicto the cell lines bearing the CD22 antigen, than was the immunotoxinthat incorporated an scFv with the wild type sequence of the V_(H)CDR3of RFB4. Since the GKNR (SEQ ID NO:18) mutant was selected because of anapparent increased binding affinity, it would be expected to be morecytotoxic when incorporated into the structure of a chimeric immunotoxinas was the case for the other mutantb scFvs. However, phage displayselects for increased expression as well as increased antibody bindingaffinity. Therefore a dot blot analysis was performed to compare therelative number of wild type (GSSY) (SEQ ID NO:13) scFvs displayed onphage to the number of the GKNR (SEQ ID NO:18) mutant scFvs displayed onphage. The dot blot indicated that the GKNR (SEQ ID NO:18) mutant scFvwas overexpressed on the phage. GKNR (SEQ ID NO:18) was not analysedfurther.

Mutant immunotoxins were not cytotoxic to the CD22 negative cell lineHUT-102, indicating that the cytotoxic effect of the immunotoxins isselective to CD22 antigen positive cells.

Thus, by incorporating the mutant scFvs with a higher binding affinityfor CD22 antigen into the structure of chimeric RFB4-PE38 immunotoxins,the cytotoxicity of the immunotoxins toward cells bearing the CD22antigen is enhanced. Therefore, the mutant cytotoxins are more effectiveat inhibiting growth of the antigen bearing cells than is theimmunotoxin with the wild-type (GSSY) (SEQ ID NO:13) V_(H)CDR3 of RBF4.

Example 3

The studies reported in this Example demonstrate that the mutantRFB4-PE38 immunotoxins of Example 2 are more effective at inhibiting thegrowth of malignant cells taken from patients with advanced lymphocyticdisease than is the chimeric immunotoxin bearing the wild-type V_(H)CDR3of RFB4, RFB4-PE38.

Cytotoxic activity of mutant immunotoxins on malignant cells isolatedfrom patients was measured in blood samples collected from patients aspart of approved clinical protocols at the NIH. Patients 1, 2, 3, and 5have chronic lymphocytic leukemia (CLL). Patient 4 has hairy cellleukemia (HCL). Samples were processed as previously described(Kreitman, R. J. et al., Clin. Cancer Res., 6:1476-1487 (2000)).Briefly, protein synthesis was measured by counting cpm of [³H] leucineincorporated into protein. Inhibition of protein synthesis of 50%,defined as being halfway between the level of incorporation of ³[H]leucine in the absence of toxin and the level of incorporation of ³[H]leucine in the presence of 10 μg/ml of cycloheximide, was determined bycapturing radiolabeled material on filter-mats, which were then countedin a Betaplate scintillation counter (Wallac).

Protein synthetic activity of Ficoll purified mononuclear cells from thefour patients with chronic lymphocytic leukemia and from the one patientwith hairy cell leukemia was determined by incubating the cells withimmunotoxins for three days then pulsing with ³H leucine for 6-8 h. Eachassay was done in triplicate. As can be seen in Table 4, all of theimmunotoxins bearing mutant scFvs were more cytotoxic than the chimericimmunotoxin bearing the wild-type RFB4-PE38 with the amino acid sequenceGSSY (SEQ ID NO:12) in the V_(H)CDR3 region.

TABLE 4 Cytotoxic activity (IC₅₀) in ng/ml of mutant immunotoxins onpatient cells Patient 1 Patient 2 Patient 3 Patient 4 Patient 5GSSY(WT) >1000 490 ± 70 34 ± 5  6.7 ± 2.3 >1000 (SEQ ID NO: 13) GTHW 29± 10 22 ± 2 1.5 ± 0.4 <1 28 ± 6 (SEQ ID NO: 14) GYNW 105 ± 48  40 ± 53.4 ± 0.7 N.D. 41 ± 2 (SEQ ID NO: 14) GTTW >1000 95 ± 9 8.5 ± 3   1.5 ±0.6 76 ± 9 (SEQ ID NO: 16) GSTY (SEQ N.D. N.D. 15 ± 2  2.1 ± 0.7 129 ±50 ID NO: 17) Ficoll-purified mononuclear cells from patients wereobtained by an approved protocol at the NIH. Cells were incubated withimmunotoxins for three days at 37° C. and pulsed with ³[H] leucine for6-8 h; protein synthesis was measured. IC₅₀s are expressed in ng/ml,standard deviations are shown. Each assay was done in triplicate.Patients 1, 2, 3, and 5 were diagnosed with CLL, patient 4 with HCLvariant. N.D.: not determined.

When tested on cells from patient 2, the chimeric immunotoxin bearingthe wild-type GSSY (SEQ ID NO:13) amino acid sequence in the V_(H)CDR3region of RFB4 had an IC₅₀ of 490 ng/ml, the GTHW (SEQ ID NO:14) mutanthad an IC₅₀ of 22 ng/ml, the GYNW (SEQ ID NO:15) had an IC₅₀ of 40ng/ml, and mutant GTTW (SEQ ID NO:16) mutant had an IC50 of 95 ng/ml.Similarly, on samples isolated from patient 5 the chimeric immunotoxinbearing the wild-type GSSY (SEQ ID NO:13) amino acid sequence in theV_(H)CDR3 of RFB4 had an IC₅₀ of >1000 ng/ml whereas the immunotoxinwith GTHW (SEQ ID NO:14) had an IC₅₀ of 28 ng/ml and the GYNW (SEQ IDNO:15) immunotoxin had an IC₅₀ of 41 ng/ml and mutant GTTW (SEQ IDNO:16) had an IC50 of 76 ng/ml.

In most of the patients, the parental immunotoxin carrying GSSY (SEQ IDNO:13) amino acid sequence in the V_(H)CDR3 region of RFB4 was not ableto inhibit protein synthesis by 50% at the concentrations tested,therefore the IC₄₀, the toxin concentration that reduced incorporationof ³H-leucine by 40%, was determined instead.

Thus, in every case tested, the chimeric immunotoxins bearing mutantscFvs, were more effective at inhibiting protein synthesis of theleukemic cells than was the chimeric immunotoxin bearing the wild-typescFv.

Example 4

The experiments reported in this example demonstrate that the chimericRFB4-PE38 immunotoxins, GTHW (SEQ ID NO:14), GYNW (SEQ ID NO:15), GTTW(SEQ ID NO:16), and GSTY (SEQ ID NO:17), which bear the mutant scFvs,bind recombinant CD22 antigen with higher affinity than the chimericRFB4-PE38 immunotoxin with the wild-type V_(H)CDR3 of RFB4 (GSSY, SEQ IDNO:13).

Binding affinity of the chimeric immunotoxins was determined by plasmonsurface resonance (Biacore). First, CD22 recombinant protein wasprepared and immobilized it on a CM5 chip. The extracellular domain ofCD22 protein was expressed as a fusion to human IgG Fc in transfected293T cells. The human Fc fragment from plasmid Ret-Fc was PCR amplifiedusing (provided by M. Billaud, Laboratoire de Genetique, Lyon, France)primers which introduced 5′ NotI and 3′ XbaI restriction sites:

(SEQ ID NO: 33) 5′-GAGTGAGTGCGGCCGCGG TGGTCGTCGTGCATCCGT (SEQ ID NO: 34)5′-TCACTCACTCTAGACGGCCGTCGCACTCATTTAC

After digestion with NotI and XbaI, the PCR product was purified andcloned into the NotI and XbaI sites of vector pcDNA1.1 to create plasmidpcDNA1.1-Fc. Next, the extra-cellular portion of CD22 pcDNA 1.1-Fc wasfused in-frame with the Fc by amplifying the CD22 extracellular domainfrom plasmid pRKm22 using the following oligomers:

(SEQ ID NO: 35) 5′-GTGAGTGAGAATTCATGCATCTCCTCGGCCCCTG (SEQ ID NO: 36)5′-TCACTCACTCGCGGCCGCTTCGCCTGCCGATGGTCTC

pRKm22 is a plasmid encoding full-length human CD22β obtained by cloningfrom a Daudi cDNA Quick clone library (Clontech). The oligomersintroduced EcoRI and NotI restriction sites, which were used to clonethe purified the PCR product into pcDNA1.1-Fc to create pcDNA1.1-22-Fc.293T cells were transfected with plasmid pcDNA1.1-22-Fc by standardCaPO₄ precipitation.

Binding kinetics of the chimeric immunotoxins were measured usingBIAcore 2000 Biosensor. CD22-Fc protein was diluted to 50 μg/ml in aminecoupling buffer and immobilized to a BIAcore sensor chip CM5. Thechimeric immunotoxins were diluted to 25 μg/ml in HEPES-buffered saline,and on and off rates were measured by injecting 50 μg of immunotoxinover the chip surface at 10 μl/min, and then allowing the bound materialto dissociate for 5 min or more. The remaining bound material wasremoved from CD22 protein by injecting 10 μl of 20 mM phosphoric acid.Each immunotoxin was injected and analyzed at least three times. Bindingkinetics were determined using BIA evaluation 2.1 software.

Comparison of the binding profiles of immunotoxins bearing the wild typeGSSY (SEQ ID NO:13) amino acid sequence in the V_(H)CDR3 region with thebinding profile of the mutant immunotoxins with GTTW (SEQ ID NO:16),GYNW (SEQ ID NO:15) and GTHW (SEQ ID NO:14) in the V_(H)CDR3 regionrevealed that these three mutant immunotoxins had slower dissociationrates than the GSSY (SEQ ID NO:13) wild-type. In some cases the mutantimmunotoxins also had faster association rates compared to wild-typeGSSY (SEQ ID NO:13)-containing immunotoxin. In every case however,overall binding affinity of the mutant chimeric immunotoxins exceededthat of the wild-type. Kd was calculated by dividing K_(off) by K_(on).The binding constants, K_(on), K_(off) and Kd are shown below in Table5.

TABLE 5 Summary of Biacore analysis of RFB4-PE38 mutants ImmunotoxinK_(on)(M⁻¹S⁻¹) K_(off)(S⁻¹) KD (nM) GSSY (WT) (SEQ 2.08 × 10⁴ 1.77 ×10⁻³ 85 ID NO: 13) GTHW (SEQ ID 3.27 × 10⁴ 2.07 × 10⁻⁴ 6 NO: 14) GYNW(SEQ ID 1.84 × 10⁴ 1.91 × 10⁻⁴ 10 NO: 15) GTTW (SEQ ID 2.62 × 10⁴  6.5 ×10⁻⁴ 24 NO: 16) GSTY (SEQ ID 3.15 × 10⁴ 1.55 × 10⁻³ 49 NO: 17)

The wild-type, GSSY (SEQ ID NO:13) immunotoxin had a Kd of 85 nM,whereas mutant with highest affinity, GTHW (SEQ ID NO:14), had a Kd of 6nM, mutant GYNW (SEQ ID NO:15) had a Kd of 10 nM and mutant GTTW (SEQ IDNO:16) had a Kd of 24 nM. Mutant GSTY (SEQ ID NO:17) had a Kd of 49 nM.

Thus, the chimeric immunotoxins bearing mutant scFvs GTHW (SEQ IDNO:14), GYNW (SEQ ID NO:15), GTTW (SEQ ID NO:16), and GSTY (SEQ IDNO:17), bind recombinant CD22 antigen with higher affinity than thechimeric immunotoxin bearing an scFv with the wild-type V_(H)CDR3 ofRFB4 through various combinations of faster association rates and slowerdissociation rates of the mutant immunotoxins relative to that of thewild-type chimeric immunotoxin.

Example 5

The studies reported in this Example show that an exemplar chimericdisulfide-stabilized (dsFv) RFB4-PE38 immunotoxin made with the GTHW(SEQ ID NO:14) sequence was strikingly more effective at inhibiting thegrowth of malignant cells taken from patients with advanced lymphocyticdisease than a like chimeric dsFv immunotoxin bearing the wild-typeV_(H)CDR3 of RFB4 (GSSY, SEQ ID NO:13).

Disulfide-stabilized Fvs of both sequences were made as previouslydescribed for making RFB4(dsFv)-PE38. See, Kreitman et al., Clin CancerRes 6(4):1476-87 (2000). See also, e.g., U.S. Pat. Nos. 5,747,654,6,147,203, 6,074,644, and 5,980,895. The immunotoxins were testedagainst cells taken from seven patients with CLL and against cells takenfrom two patients with HCL. Cytotoxicity assays were performed as setforth in Example 3, above.

As shown in Table 6, dsFv immunotoxin made with the GTHW (SEQ ID NO:14)sequence was from 10 to 40 times more cytotoxic to cells from patientswith CLL than was dsFv immunotoxin made with the wildtype RFB4 sequence.Similarly, the dsFv immunotoxin made with the GTHW (SEQ ID NO:14)sequence was from 4 to 7 times more cytotoxic to cells from patientswith HCL than was the dsFv immunotoxin made from the wildtype RFB4sequence. Thus, dsFv immunotoxins made with the mutated RFB4 sequencesof the invention demonstrate strikingly higher cytotoxicity to cellsfrom patients with advanced lymphocytic disease than dsFv immunotoxinsmade with the wild-type RFB4 sequence.

TABLE 6 Improved Cytotoxic Activity of GTHW (SEQ ID NO: 14) Mutant dsFvImmunotoxin Towards Cells from Patients with CLL or HCL IC₅₀ (ng/ml) Pt.GTHW (SEQ Fold No. Disease WT ID NO: 14) Improvement 1 CLL 41 1.8 23 2CLL 128 11.2 11 3 CLL 220 22.0 10 4 CLL 49 4.6 11 5 CLL 172 6.4 27 6CLL >1000 25 >40 7 CLL 119 5.8 20 8 HCLv 4 0.54 7 9 HCL 6 1.6 4

While specific examples have been provided, the above description isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

All publications and patent documents cited herein are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent document were so individuallydenoted. Citation of various references in this document is not anadmission that any particular reference is considered to be “prior art”to the invention.

1. An isolated anti-CD22 antibody with a variable light (V_(L)) chainhaving the sequence of the V_(L) chain of antibody RFB4 and a variableheavy (V_(H)) chain having the sequence of the V_(H) chain of antibodyRFB4, provided that residues 100, 100A and 100B of CDR3 of the V_(H)chain of said anti-CD22 antibody, as the residues of the V_(H) chain arenumbered in the column of FIG. 3 according to “Kabat Numbering System”,have an amino acid sequence selected from the group consisting of: THW,YNW, TTW, and STY.
 2. An antibody of claim 1, wherein said antibody isselected from the group consisting of an scFv, a dsFv, a Fab, or aF(ab′)₂.
 3. A composition comprising an antibody of claim 1 conjugatedor fused to a therapeutic moiety or a detectable label.
 4. A compositionof claim 3, wherein the therapeutic moiety is selected from the groupconsisting of a cytotoxin, a drug, a radioisotope, or a liposome loadedwith a drug or a cytotoxin.
 5. A composition of claim 4, wherein thetherapeutic moiety is a cytotoxin.
 6. A composition of claim 5, whereinthe cytotoxin is selected from the group consisting of ricin A, abrin,ribotoxin, ribonuclease, saporin, calicheamycin, diphtheria toxin or acytotoxic subunit or mutant thereof, a Pseudomonas exotoxin, a cytotoxicportion thereof, a mutated Pseudomonas exotoxin, a cytotoxic portionthereof, and botulinum toxins A through F.
 7. A composition of claim 6,wherein said cytotoxin is a Pseudomonas exotoxin or cytotoxic fragmentthereof, or a mutated Pseudomonas exotoxin or a cytotoxic fragmentthereof.
 8. A composition of claim 7, wherein said Pseudomonas exotoxinis selected from the group consisting of PE35, PE38, PE38KDEL, PE40,PE4E, and PE38QQR.
 9. A composition of claim 8, wherein the Pseudomonasexotoxin is PE38.
 10. A composition of claim 3, further comprising apharmaceutically acceptable carrier.
 11. A composition of claim 4,further comprising a pharmaceutically acceptable carrier.
 12. Acomposition of claim 5, further comprising a pharmaceutically acceptablecarrier.
 13. A composition of claim 6, further comprising apharmaceutically acceptable carrier.
 14. A composition of claim 7,further comprising a pharmaceutically acceptable carrier.
 15. Acomposition of claim 8, further comprising a pharmaceutically acceptablecarrier.
 16. A composition of claim 9, further comprising apharmaceutically acceptable carrier.
 17. A method of inhibiting growthof a CD22+ cancer cell by contacting said cell with an anti-CD22antibody with a variable light (V_(L)) chain having the sequence of aV_(L) of antibody RFB4 and a variable heavy (V_(H)) chain having thesequence a V_(H) chain of antibody RFB4, provided that residues 100,100A and 100B of CDR3 of the V_(H) chain of said anti-CD22 antibody, asthe residues are numbered in the column of FIG. 3 according to “KabatNumbering System”, have an amino acid sequence selected from the groupconsisting of: THW, YNW, TTW, and STY, which antibody is fused orconjugated to a therapeutic moiety, which therapeutic moiety inhibitsgrowth of said cell.
 18. A method of claim 17, wherein said antibody isselected from the group consisting of an scFv, a dsFv, a Fab, or aF(ab′)₂.
 19. A method of claim 17, wherein said therapeutic moiety isselected from the group consisting of a cytotoxin, a drug, aradioisotope, or a liposome loaded with a drug or a cytotoxin.
 20. Amethod of claim 19, wherein the therapeutic moiety is a cytotoxin.
 21. Amethod of claim 19, wherein the cytotoxin is selected from the groupconsisting of ricin A, abrin, ribotoxin, ribonuclease, saporin,calicheamycin, diphtheria toxin or a cytotoxic subunit or mutantthereof, a Pseudomonas exotoxin, a cytotoxic portion thereof, a mutatedPseudomonas exotoxin, a cytotoxic portion thereof, and botulinum toxinsA through F.
 22. A method of claim 21, wherein said cytotoxin is aPseudomonas exotoxin or cytotoxic fragment thereof, or a mutatedPseudomonas exotoxin or a cytotoxic fragment thereof.
 23. A method ofclaim 22, wherein said Pseudomonas exotoxin is selected from the groupconsisting of PE35, PE38, PE38KDEL, PE40, PE4E, and PE38QQR.
 24. Amethod of claim 23, wherein the Pseudomonas exotoxin is PE38.
 25. Amethod for detecting the presence of a CD22+ cancer cell in a biologicalsample, said method comprising: (a) contacting cells of said biologicalsample with an anti-CD22 antibody with a variable light (V_(L)) chainhaving the sequence of a V_(L) chain of antibody RFB4 and a variableheavy (V_(H)) chain having the sequence of a V_(H) chain of antibodyRFB4, provided that residues 100, 100A and 100B of CDR3 of the V_(H)chain of said anti-CD22 antibody, as the residues are numbered in thecolumn of FIG. 3 according to “Kabat Numbering System”, have an aminoacid sequence selected from the group consisting of: THW, YNW, TTW, andSTY, said antibody being fused or conjugated to a detectable label; and,(b) detecting the presence or absence of said label, wherein detectingthe presence of said label indicates the presence of a CD22+ cancer cellin said sample.
 26. A method of claim 25, wherein said antibody isselected from the group consisting of an scFv, a dsFv, a Fab, or aF(ab′)₂.
 27. A kit for detecting the presence of a CD22+ cancer cell ina biological sample, said kit comprising: (a) a container, and (b) ananti-CD22 antibody with a variable light (V_(L)) chain having thesequence of a V_(L) chain of antibody RFB4 and a variable heavy (V_(H))chain having the sequence of a V_(H) chain of antibody RFB4, providedthat residues 100, 100A and 100B of CDR3 of the V_(H) chain of saidanti-CD22 antibody, as the residues are numbered in the column of FIG. 3according to “Kabat Numbering System”, have an amino acid sequenceselected from the group consisting of: THW, YNW, TTW, and STY, whichantibody is fused or conjugated to a detectable label.
 28. A kit ofclaim 27, wherein said antibody is selected from the group consisting ofan scFv, a dsFv, a Fab, or a F(ab′)₂.